The molecular and cellular mechanisms of synaptic specificity, i.e. the establishment of synapses between correct partners, have largely been studied through guidance receptors. These receptors are expressed on the surface of growth cones and mediate attraction or repulsion. It has become increasingly clear that most guidance receptors are not constitutively present on the cell surface, but instead exhibit spatiotemporally dynamic localization. In many cases, overexpression of guidance receptors causes worse defects than their loss. Hence, it is not only the combinatorial expression of guidance receptors that determines synaptic specificity: Guidance receptors must be presented at the right time and place on the membrane to convey meaningful synapse formation signals. Our working hypothesis is that developmental vesicle trafficking plays instructive roles during the synaptic specification process by regulating the dynamic localization of guidance receptors. While there is a wealth of literature on the effect of loss- and gain-of- function of guidance receptors on synaptic specificity, few studies address how their dynamic localization is regulated. In the proposed research, we will combine genetic approaches with live imaging to systematically address this gap. Specifically, we will utilize unique advantages of the Drosophila visual system to reveal the function of intracellular trafficking in the establishment of synaptic specificity. In Specific Aim 1, we will identify the implicated vesicle trafficking compartments based on systematic profiling of fluorescently tagged Rab proteins, small GTPases that are key regulators of intracellular trafficking. So far, we have identified at least one novel and Rab19-dependent trafficking pathway required for guidance receptor trafficking in visual development. In Specific Aim 2 we will therefore focus on a detailed characterization of Rab19. In addition, we have identified an intracellular trafficking pathway that depends on the vesicular ATPase. The vesicular ATPase is known to regulate protein trafficking via acidification of intracellular compartments. We have found that this pathway is cell-specifically required in optic lobe interneurons, but not photoreceptors, for guidance receptor trafficking during visual development. In Specific Aim 3 we will elucidate the cell-specific trafficking mechanisms by uncovering the different functions of the vesicular ATPase in photoreceptors and optic lobe interneurons. In summary, the proposed research will identify novel mechanisms that direct the spatiotemporal regulation of guidance receptors and characterize their contribution to synaptic specificity in the visual system. PUBLIC HEALTH RELEVANCE: Intracellular vesicle trafficking is fundamental to most aspects of cellular development and function; its disruption can cause severe diseases, e.g. Griscelli syndrome, Charcot-Marie-Tooth type 2B disease, Warburg Micro Syndrome, X-linked mental retardation and progressive retinal degeneration (choroideraemia). Importantly, many of these diseases affect brain development and function. The experiments in this proposal are designed to systematically fill gaps in our understanding of intracellular vesicle trafficking during brain development using the Drosophila visual system as a model. [unreadable] [unreadable]